The present invention relates to a color translating microscope employing ultraviolet light in place of or in addition to visible and/or infrared light sources. More specifically, the present invention relates to a method and a microscope which determine and represent differential absorption, transmission, reflection, fluorescent and/or Raman characteristics of a sample as a color image to a user.
It has been desired for some time to find a low cost, reliable and yet flexible means to view living and/or dynamic processes at high resolution in real time. Another desire is to be able to carry out wide ranging spectral imaging based on differential spectral absorption after such as, Caspersson, T., 1940, xe2x80x9cMethods for the determination of the absorption spectra of cell structuresxe2x80x9d, Journal of the Royal Microscopical Society, 60, 8-25, to study biological samples without the addition of any contrast media. Yet another desire is to substantially reduce the amount of light that can potentially damage or affect the behaviour of a sample. In other words, the desire has been to view a sample with the slightest possible interference with its normal behaviour in order to see its operation in a state substantially the same as that which it would normally experience in its usual environment. Accordingly, it has been desired to eliminate stains, fluorochromes, dyes, fixatives, preservatives or other additives and to minimize external fields and radiations such as magnetic, electrical or photon energy.
Color translating UV microscopes are known. In the past many inventors have attempted to produce color translating UV microscopes. For example, some prior art microscopes have used photographic techniques as described in: Barnard, J. E., 1919, xe2x80x9cThe limitations of microscopyxe2x80x9d, Journal of the Royal Microscopical Society, 39, 1-13; Martin, L. C., Johnson. 1928, B. K., xe2x80x9cUV Microscopyxe2x80x9d, parts 1 and 2, Journal of Scientific Instruments, 5, 337-344 and 380-387; Lucas, F. F., 1930, xe2x80x9cThe architecture of living cellsxe2x80x9d, Proceedings of the National Academy of Sciences, 16, 599-607; Barnard, J. E., 1939, xe2x80x9cTowards the smallest living thingsxe2x80x9d, Journal of the Royal Microscopical Society, 59, 1-10; Brumberg, E. M., 1946, xe2x80x9cA microscope for visual colour microscopy in the ultraviolet raysxe2x80x9d, Comptes Rendus (Doklady) de I""Academie des Sciences de I""URSS, 52:6, 499-502; and Land, E. H., et al, 1949, xe2x80x9cA colour translating UV microscopexe2x80x9d, Science, 109, 371-374. The contents of these publications are incorporated herein by reference.
Other prior art attempts at color translating UV microscopes have been made using video techniques as described in: Zworykin, V. K., Hatke, F. L., 1957, xe2x80x9cUltraviolet television colour translating microscopexe2x80x9d, Science, 126, 805-810; Zworykin, V. K., Berkley, C., 1962, xe2x80x9cUltraviolet colour translating television microscopyxe2x80x9d, Annals of the New York Academy of Science, 97, 364-379; Caspersson, T., 1964, xe2x80x9cThe ultraviolet microscopexe2x80x9d, Journal of the Royal Microscopical Society, 83, 67-68; and Caspersson, T., 1964, xe2x80x9cThe study of living cells with the ultraviolet microscopexe2x80x9d, Journal of the Royal Microscopical Society, 83, 95-96. The contents of these publications are incorporated herein by reference.
It is believed that all these prior art attempts failed due to the complex nature of the solutions attempted, the attendant costs and the high operating and maintenance burden and costs. The results from these systems were mediocre at best due to the delay in image availability in the photographic processes and due to the low resolution and long integration times of the video solutions available at the time the work was carried out.
A more recent attempt at a useful UV microscope is shown in U.S. Pat. No. 5,481,401 to Kita et al., the contents of which are incorporated herein by reference. As shown in FIG. 9 of this reference, a final image is produced from the combination of a monochromatic UV microscope image with a color visible light image to obtain a pseudo color image. In other embodiments taught by the reference, separate displays of the monochromatic UV image and the color visible light image are provided to the user. This reference suffers from disadvantages in that, for example, it needs high power UV illumination to provide sufficient illumination to the UV video camera which will be detrimental to the sample, it does not combine multiple three UV images from the same camera created with successive selections of light of different wavelength center and bandpass to create a full three colour visible image and therefore it is prone to misalignment of the individual cameras, and it is preset and not rapidly adjustable as to the wavelengths of light chosen for imaging, it does not use the extending resolving power of the deep UV range of the spectrum in which cellular absorption of biological specimens begins to offer the advantages of absorption staining of living systems and it will not resolve images at resolutions greater than those possible under visible light viewing conditions, as the final displayed visible light and monochromatic UV images are presented to the user at the same pixel resolution.
It is desired to have a color translating UV microscope which provides substantially real time image presentation without damage to the sample and which ranges from the relatively simple to construct and to use simple version to the powerful and comprehensive imaging system in the research version described herein.
It is an object of the present invention to provide a novel color translating UV microscope which obviates or mitigates at least one disadvantage of the prior art.
According to a first aspect of the present invention, there is provided a microscope for translating spectral information to a visible color image in which light from a source is separated into components by either a set of two or more filters or a device for providing wavelength limited light and then passed through or reflected off the sample and then imaged by an objective lens onto a video camera where it is converted to visible light by a fluorescent coating on the photosensitive surface of video camera which provides the image as an electronic signal which is then converted into electronic data by a video to computer interface system and then recombined into a multicolor image by computer processing finally creating a color visible image on a display monitor where the computer is supplied with information on the position of the filters or wavelength limited light in order to synchronize acquisition of the images and the color translation and recombination process.
According to another aspect of the present invention, there is provided a microscope for translating spectral information to a visible color image in which light from one or more sources is separated into components by either a set of two or more filters or device for providing wavelength limited light and then passed through or reflected off an sample then imaged onto the input of an image intensifier by an objective lens then converted to visible light by the image intensifier or other wavelength translating device the output of which is then imaged on the input of a video camera which provides the image as an electronic signal which is then converted into electronic data by a video to computer interface system and then recombined into a multicolor image by computer processing finally creating a color visible image on a display monitor where the computer is supplied with information on the position of the filters or wavelength limited light in order to synchronize acquisition of the images and the color translation and recombination process.
According to yet another aspect of the present invention, there is provided a microscope for translating spectral information to a visible color image in which light from a source which emits narrow spectral lines, as opposed to a continuum of spectra, is separated into components after passing through a sample and is then converted to visible polychromatic light by a converter such as an image intensifier and is then recombined into a multicolor image by a combining images captured by a video camera, video interface and computer where such images are synchronized with the filter system.
According to yet another aspect of the present invention, there is provided an optical microscope system where an image intensifier and CCD camera combined with a computerized image capture and image processing system is used to convert images collected in wavelengths outside the normal range of human vision, such as soft x-ray, UV or IR, to visible images and where, while at least one of the images collected is in the range 200 nanometers to 300 nanometers, some of the other images used to produce the final color image can be collected in the range from 300 to 3300 nanometers.
According to yet another aspect of the present invention, there is provided a microscope which includes active optical feedback for stabilization of the position and intensity of the illuminating optical system.
According to yet another aspect of the present invention, there is provided a microscope which includes active optical monitoring for recording and providing the data to allow relating the effects of the dosage of the illuminating radiation to the observed effects in the samples and for modulation of that illumination to prolong sample life.
According to yet another aspect of the present invention, there is provided a microscope that is capable of selecting between brightfield, darkfield, and reflected brightfield or reflected darkfield illumination or phase contrast or other standard forms of illumination under computer control.
According to yet another aspect of the present invention, there is provided a microscope that is capable of switching objective lenses under computer control.
According to yet another aspect of the present invention, there is provided a microscope that is capable of switching image intensifiers under computer control.
According to yet another aspect of the present invention, there is provided a microscope that is capable of switching video cameras under computer control.
It is an object of yet another embodiment of the present invention to provide a novel color translating microscope which obviates or mitigates at least one of the difficulties of the prior art. It is a further object to provide a novel method of forming a color image of the differential absorption of a microscope sample.
According to yet another aspect of the present invention, there is provided a microscope for translating spectral information to a visible colour image in which light from a source is separated into components by a set of two or more filters then passed through an sample then converted to visible polychromatic light by a converter such as an image intensifier and then recombined into a multicolour image by a set of two or more filters where such filter sets are synchronized with each other.
According to yet another aspect of the present invention, there is provided a method of producing an image representing the differential absorption of light by a sample, comprising the steps of:
(i) illuminating a sample with light of a first desired wavelength by imposing an illumination filter between a multiwavelength light source and the sample;
(ii) receiving light from the sample at a photon gain device which converts the received light to an intensified white light;
(iii) filtering said intensified white light with an image filter to obtain visible light at preselected wavelength for said desired wavelength;
(iv) forming an image of said filtered intensified white light; and
(v) synchronously changing said illumination filter and said image filter and repeating steps (i) through (iv) to illuminate said sample with light of a second desired wavelength and to form an image from visible light obtained from said intensified white light at a second preselected wavelength for said second desired wavelength.